Apparatus and method for maintaining and/or restoring viability of organs

ABSTRACT

An organ perfusion apparatus and method monitor, sustain and/or restore viability of organs and preserve organs for storage and/or transport. The method includes perfusing the organ at hypothermic and/or normothermic temperatures, preferably after hypothermic organ flushing for organ transport and/or storage. The method can be practiced with prior or subsequent static or perfusion hypothermic exposure of the organ. Organ viability is restored by restoring high energy nucleotide (e.g., ATP) levels by perfusing the organ with a medical fluid, such as an oxygenated cross-linked hemoglobin-based bicarbonate medical fluid, at normothermic temperatures. In perfusion, organ perfusion pressure is preferably controlled in response to a sensor disposed in an end of tubing placed in the organ, by a pneumatically pressurized medical fluid reservoir, providing perfusion pressure fine tuning, overpressurization prevention and emergency flow cut-off. In the hypothermic mode, the organ is perfused with a medical fluid, preferably a simple crystalloid solution containing antioxidants, intermittently or in slow continuous flow. The medical fluid may be fed into the organ from an intermediary tank having a low pressure head to avoid organ overpressurization. Preventing overpressurization prevents or reduces damage to vascular endothelial lining and to organ tissue in general. Viability of the organ may be automatically monitored, preferably by monitoring characteristics of the medical fluid perfusate. The perfusion process can be automatically controlled using a control program.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/537,180,filed Mar. 29, 2000, which is a continuation-in-part of application Ser.No. 09/162,128, filed Sep. 29, 1998, the entire contents of which arehereby incorporated by reference.

The invention relates to an apparatus and method for perfusing one ormore organs to monitor, sustain and/or restore the viability of theorgan(s) and/or for transporting and/or storing the organ(s).

BACKGROUND

Preservation of organs by machine perfusion has been accomplished athypothermic temperatures with or without computer control withcrystalloid perfusates and without oxygenation. See, for example, U.S.Pat. Nos. 5,149,321, 5,395,314, 5,584,804, 5,709,654 and 5,752,929 andU.S. patent application Ser. No. 08/484,601 to Klatz et al., which arehereby incorporated by reference. Hypothermic temperatures provide adecrease in organ metabolism, lower the energy requirements, delay thedepletion of high energy phosphate reserves and accumulation of lacticacid and retard the morphological and functional deteriorationassociated with disruption of blood supply. Oxygen can not be utilizedefficiently by mitochondria below approximately 20° C. to produce energyand the reduction in catalase/superoxide dismutase production andascorbyl and glutathione regeneration at low temperatures allows highfree radical formation. The removal of oxygen from perfusates during lowtemperature machine perfusion has even proven helpful in improving organtransplant results by some investigators.

Reduction in potential oxygen damage is also accomplished via theaddition of antioxidants to the perfusate. In particular, this hasproven useful in reducing organ damage after long warm ischemia times.Numerous other perfusate additives have also been reported to improvethe outcome of machine perfusion.

Ideally organs would be procured in a manner which limits their warmischemia time to essentially zero. Unfortunately, in reality, manyorgans, especially from non-beating heart donors, are procured afterextended warm ischemia time periods (i.e. 45 minutes or more). Themachine perfusion of these organs at low temperature has demonstratedsignificant improvement (Transpl Int 1996 Daemen). Further, prior artteaches that the low temperature machine perfusion of organs ispreferred at low pressures (Transpl. Int 1996 Yland) with roller ordiaphragm pumps delivering the perfusate at a controlled pressure.Numerous control circuits and pumping configurations have been utilizedto achieve this objective and to machine perfuse organs in general. See,for example, U.S. Pat. Nos. 5,338,662 and 5,494,822 to Sadri; U.S. Pat.No. 4,745,759 to Bauer et al.; U.S. Pat. Nos. 5,217,860 and 5,472,876 toFahy et al.; U.S. Pat. No. 5,051,352 to Martindale et al.; U.S. Pat. No.3,995,444 to Clark et al.; U.S. Pat. No. 4,629,686 to Gruenberg; U.S.Pat. Nos. 3,738,914 and 3,892,628 to Thome et al.; U.S. Pat. Nos.5,285,657 and 5,476,763 to Bacchi et al.; U.S. Pat. No. 5,157,930 toMcGhee et al.; and U.S. Pat. No. 5,141,847 to Sugimachi et al. However,the use of such pumps for machine perfusion of organs increases the riskof overpressurization of the organ should the organ perfusion apparatusmalfunction. High pressure perfusion (e.g., above about 60 mm Hg) canwash off the vascular endothelial lining of the organ and in generaldamages organ tissue, in particular at hypothermic temperatures wherethe organ does not have the neurological or endocrinal connections toprotect itself by dilating its vasculature under high pressure.

Furthermore, the techniques used for assessment of the viability ofthese machine perfused organs have been a critical factor in limitingthe organs from greater use. While increased organ resistance (i.e.,pressure/flow) measurements during machine perfusion are a usefulindicator, they demonstrate only the worst case situations.

During the low temperature machine perfusion of organs which have beendamaged by warm ischemia time or by the machine perfusion itself, theorgans will elute intracellular and endothelial as well as membraneconstituents. Over the years the appearance of various ubiquitousintracellular enzymes, such as lactic dehydrogenase (LDH) and alkalinephosphatase, in the perfusate has been used as a biomarker of organdamage. Recently, the determination of the presence of alphaglutathione-S-transferase (a-GST) and Pi glutathione-S-transferase(p-GST) in low temperature machine perfusion perfusates has proven asatisfactory indicator in predicting the functional outcome ofnon-beating heart donor kidney grafts before transplantation (Transpl1997 Daemen).

The prior art has also addressed the need to restore or maintain anorgan's physiological function after preservation for an extended periodof time at hypothermic temperatures. In particular, U.S. Pat. No.5,066,578 to Wikman-Coffelt discloses an organ preservation solutionthat contains large amounts of pyruvate. Wikman-Coffelt teaches thatflooding of the organ with pyruvate bypasses glycosis, the step in thecell energy cycle that utilizes adenosine triphosphate (ATP) to producepyruvate, and pyruvate is then available to the mitochondria foroxidative phosphorylation producing ATP. Wikman-Coffelt teachesperfusing or washing an organ at a warm temperature with a firstpreservation solution containing pyruvate for removal of blood or otherdebris from the organ's vessels and to vasodilate, increase flow andload the cells with an energy supply in the form of a clean substrate,namely the pyruvate. Wikman-Coffelt teaches that the pyruvate preventsedema, ischemia, calcium overload and acidosis as well as helps preservethe action potential across the cell membrane. The organ is thenperfused with a second perfusion solution containing pyruvate and asmall percentage of ethanol in order to stop the organ from working,vasodilate the blood vessels allowing for full vascular flow, continueto load the cells with pyruvate and preserve the energy state of theorgan. Finally the organ is stored in a large volume of the firstsolution for 24 hours or longer at temperatures between 4° C. and 10° C.

However, the mitochondria are the source of energy in cells and needsignificant amounts of oxygen to function. Organs naturally havesignificant pyruvate levels, and providing an organ with additionalpyruvate will not assist in restoring and/or maintaining an organ's fullphysiological function if the mitochondria are not provided withsufficient oxygen to function. Further, briefly flooding an organ withpyruvate may, in fact, facilitate tearing off of the vascularendothelial lining of the organ.

U.S. Pat. No. 5,599,659 to Brasile et al. also discloses a preservationsolution for warm preservation of tissues, explants, organs andendothelial cells. Brasile et al. teach disadvantages of cold organstorage, and proposed warm preservation technology as an alternative.Brasile et al. teach that the solution has an enhanced ability to serveas a medium for the culture of vascular endothelium of tissue, and as asolution for organs for transplantation using a warm preservationtechnology because it is supplemented with serum albumin as a source ofprotein and colloid; trace elements to potentiate viability and cellularfunction; pyruvate and adenosine for oxidative phosphorylation support;transferrin as an attachment factor; insulin and sugars for metabolicsupport and glutathione to scavenge toxic free radicals as well as asource of impermeant; cyclodextrin as a source of impermeant, scavenger,and potentiator of cell attachment and growth factors; a high Mg++concentration for microvessel metabolism support; mucopolysaccharides,comprising primarily chondroitin sulfates and heparin sulfates, forgrowth factor potentiation and hemostasis; and ENDO GRO™ as a source ofcooloid, impermeant and specific vascular growth promoters. Brasile etal. further teach warm perfusing an organ for up to 12 hours at 30° C.,or merely storing the organ at temperatures of 25° C. in thepreservation solution.

However, flooding an organ with such chemicals is insufficient to arrestor repair ischemic injury where the mitochondria are not provided withsufficient oxygen to function to produce energy. The oxygen needs of anorgan at more than 20° C. are substantial and cannot be met by a simplecrystalloid at reasonable flows. Further, assessment of the viability ofan organ is necessary before the use of any type of solution can bedetermined to have been fruitful.

SUMMARY

The present invention focuses on avoiding damage to the organ duringperfusion while monitoring, sustaining and/or restoring the viability ofthe organ and preserving the organ for storage and/or transport. Theinvention is directed to an apparatus and method for perfusing an organto monitor, sustain and/or restore the viability of the organ and/or fortransporting and/or storing the organ. More particularly, the organperfusion apparatus and method according to the invention monitor,sustain and/or restore organ viability by perfusing the organ athypothermic temperature (hypothermic perfusion mode) and/or normothermictemperatures (normothermic perfusion mode) preferably after flushing ofthe organ such as by hypothermic flushing followed by static organstorage and/or organ perfusion at hypothermic temperatures for transportand/or storage of the organ.

The restoring of organ viability may be accomplished by restoring highenergy nucleotide (e.g., adenosine triphosphate (ATP)) levels and enzymelevels in the organ which were reduced by warm ischemia time and/orhypoxia by perfusing the organ with an oxygenated medical fluid, such asan oxygenated cross-linked hemoglobin-based bicarbonate medical fluid,at normothermic or near-normothermic temperatures. The organ may beflushed with a medical fluid prior to perfusion with the oxygenatedmedical fluid. Such perfusion can be performed at either normothermic orhypothermic temperatures, preferably at hypothermic temperatures. Forhypothermic flush, static storage and hypothermic perfusion, the medicalfluid preferably contains little or no oxygen and preferably includesantioxidants, both molecular (e.g., 2-ascorbic acid tocopherol) andenzymatic (e.g., catalase and superoxide dismutase (SOD)). Normothermicand/or hypothermic perfusion, and preferably hypothermic perfusion, canbe performed in vivo as well as in vitro. Such perfusion arrestsischemic injury in preparation for transport, storage and/or transplantof the organ.

The normothermic treatment is preferably employed after an organ thathas been subjected to hypothermic temperatures, statically and/or underperfusion. Such initial hypothermic exposure can occur, for example,during transport and/or storage of an organ after harvesting. Thetreatment is also suitable for organs that will ultimately be storedand/or transported under hypothermic conditions. In other words, thetreatment can be applied to organs prior to cold storage and/ortransport.

In the normothermic perfusion mode, gross organ perfusion pressure ispreferably provided by a pneumatically pressurized medical fluidreservoir controlled in response to a sensor disposed in an end oftubing placed in the organ, which may be used in combination with astepping motor/cam valve or pinch valve which provides for perfusionpressure fine tuning, prevents overpressurization and/or providesemergency flow cut-off. Substantially eliminating overpressurizationprevents and/or reduces damage to the vascular endothelial lining and tothe organ tissue in general. Viability of the organ may be monitored,preferably automatically, in the normothermic perfusion mode, preferablyby monitoring organ resistance (pressure/flow) and/or pH, pO₂, pCO₂,LDH, T/GST, Tprotein, and fluorescent tagged copolymer levels in themedical fluid that has been perfused through the organ and collected.

Normothermic perfusion may be preceded by and/or followed by hypothermicperfusion. In the hypothermic mode, the organ is perfused with a medicalfluid containing substantially no oxygen, preferably a simplecrystalloid solution preferably augmented with antioxidants,intermittently or at a slow continuous flow rate. Hypothermic perfusionalso can be performed in vivo as well as in vitro prior to removal ofthe organ from the donor. Hypothermic perfusion reduces the organ'smetabolic rate allowing the organ to be preserved for extended periodsof time. The medical fluid is preferably fed into the organ by pressurefrom an intermediary tank which has a low pressure head sooverpressurization of the organ is avoided. Alternatively, inembodiments, gravity can be used to feed the medical fluid into theorgan from the intermediary tank, if appropriate. Substantiallyeliminating overpressurization prevents or reduces damage to thevascular endothelial lining of the organ and to the organ tissue ingeneral, in particular at hypothermic temperatures when the organ hasless ability to protect itself by vascular constriction. Viability ofthe organ may also be monitored, preferably automatically, during therecovery process, preferably by monitoring organ resistance(pressure/flow) and/or pH, pO₂, pCO₂, LDH, T/GST, Tprotein, andfluorescent tagged copolymer levels in the medical fluid that has beenperfused through the organ and collected.

This invention includes a control system for automatically controllingperfusion of one or more organs by selecting between perfusion modes andcontrol parameters. Automatic perfusion may be based on sensedconditions in the system or manually input parameters. The system may bepreprogrammed or programmed during use. Default values and viabilitychecks are utilized.

The present invention also provides an organ cassette which allows anorgan to be easily and safely moved between apparatus for perfusing,storing and/or transporting the organ.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects and advantages of the invention will becomeapparent from the following detailed description of embodiments whentaken in conjunction with the accompanying drawings, in which:

FIG. 1 is an organ perfusion apparatus according to the invention;

FIG. 2 is a schematic diagram of the apparatus of FIG. 1;

FIG. 3 is a diagram of the electronics of the apparatus of FIG. 1;

FIG. 4 is an exploded view of a first pump module of a combined pump,filtration, oxygenation and/or debubbler apparatus according to theinvention;

FIG. 5 is an exploded view of a filtration module of a combined pump,filtration, oxygenation and/or debubbler apparatus according to theinvention;

FIG. 6 is an exploded view of an oxygenation module of a combined pump,filtration, oxygenation and/or debubbler apparatus according to theinvention;

FIG. 7 is an exploded view of a debubbler module of a combined pump,filtration, oxygenation and/or debubbler apparatus according to theinvention;

FIG. 8 is an exploded view of a second pump module of a combined pump,filtration, oxygenation and/or debubbler apparatus according to theinvention;

FIG. 9 is an exploded perspective view showing the modules of FIGS. 4-8assembled together;

FIG. 10 is a front perspective view of an assembled modular combinedpump, filtration, oxygenation and/or debubbler apparatus according tothe invention;

FIGS. 11A-11D show side perspective views of various embodiments of anorgan cassette according to the invention;

FIG. 12 is an organ perfusion apparatus configured to simultaneouslyperfuse multiple organs;

FIGS. 13A and 13B show a stepping motor/cam valve according to theinvention;

FIGS. 14A-14F show another stepping motor/cam valve according to theinvention;

FIG. 15 shows a block diagram that schematically illustrates the controlsystem according to the invention; and

FIG. 16 shows an exemplary diagram of possible processing stepsaccording to the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

For a general understanding of the features of the invention, referenceis made to the drawings. In the drawings, like reference numerals havebeen used throughout to designate like elements.

FIG. 1 shows an organ perfusion apparatus 1 according to the invention.FIG. 2 is a schematic illustration of the apparatus of FIG. 1. Theapparatus 1 is preferably at least partially microprocessor controlled,and pneumatically actuated. The microprocessor 150 connection to thesensors, valves, thermoelectric units and pumps of the apparatus 1 isschematically shown in FIG. 3.

The organ perfusion apparatus 1 is capable of perfusing one or moreorgans simultaneously, at both normothermic and hypothermic temperatures(hereinafter, normothermic and hypothermic perfusion modes). All medicalfluid contact surfaces are preferably formed of or coated with materialscompatible with the medical fluid used, more preferably non-thrombogenicmaterials. As shown in FIG. 1, the apparatus 1 includes a housing 2which includes front cover 4, which is preferably translucent, and areservoir access door 3. The apparatus preferably has one or morecontrol and display areas 5 a, 5 b, 5 c, 5 d for monitoring andcontrolling perfusion.

As schematically shown in FIG. 2, enclosed within the housing 2 is areservoir 10 which preferably includes three reservoir tanks 15 a, 15 b,17. Two of the reservoir tanks 15 a, 15 b are preferably standard oneliter infusion bags, each with a respective pressure cuff 16 a, 16 b. Apressure source 20 can be provided for pressurizing the pressure cuffs16 a, 16 b. The pressure source 20 is preferably pneumatic and may be anon board compressor unit 21 supplying at least 10 LPM external cuffactivation via gas tubes 26,26 a,26 b, as shown in FIG. 2. Theinvention, however, is not limited to use of an on board compressor unitas any adequate pressure source can be employed, for example, acompressed gas (e.g., air, CO₂, oxygen, nitrogen, etc.) tank (not shown)preferably with a tank volume of 1.5 liters at 100 psi or greater forinternal pressurization. Alternatively, an internally pressurizedreservoir tank (not shown) may be used.

Gas valves 22-23 are provided on the gas tube 26 to allow for control ofthe pressure provided by the onboard compressor unit 21. Anti-back flowvalves 24 a, 24 b may be provided respectively on the gas tubes 26 a, 26b. Pressure sensors P5, P6 may be provided respectively on the gas tubes26 a, 26 b to relay conditions therein to the microprocessor 150, shownin FIG. 3. Gas valves GV₁ and GV₂ may be provided to release pressurefrom the cuffs 16 a, 16 b. One or both of gas valves GV₁ and GV₂ may bevented to the atmosphere. Gas valve GV₄ in communication with reservoirtanks 15 a, 15 b via tubing 18 a, 18 b may be provided to vent air fromthe reservoir tanks 15 a, 15 b through tubing 18. The third reservoirtank 17 is preferably pressurized by pressure released from one of thepressure cuffs via gas valve GV₂.

The medical fluid may, for example, be a simple crystalloid solution, ormay be augmented with an appropriate oxygen carrier. The oxygen carriermay, for example, be washed, stabilized red blood cells, cross-linkedhemoglobin, pegolated hemoglobin or fluorocarbon based emulsions. Themedical fluid may also contain antioxidants known to reduce peroxidationor free radical damage in the physiological environment and specificagents known to aid in tissue protection. As discussed in detail below,an oxygenated (e.g., cross-linked hemoglobin-based bicarbonate) solutionis preferred for the normothermic mode while a non-oxygenated (e.g.,simple crystalloid solution preferably augmented with antioxidants) ispreferred for the hypothermic mode. The specific medical fluids used inboth the normothermic and hypothermic modes are designed to prevent thewashing away of or damage to the vascular endothelial lining of theorgan. For the hypothermic perfusion mode, as well as for flush and/orstatic storage, a preferred solution is the solution disclosed in U.S.Provisional Patent Application No. 60/179,153, filed Jan. 31, 2000, theentire disclosure of which is incorporated herein by reference. Ofcourse, other suitable solutions and materials may be used, as is knownin the art.

he perfusion solution may be provided in a perfusion solution kit, forexample, a saleable package preferably containing at least one firstcontainer holding a first perfusion solution for normothermic perfusionand at least one second container holding a second, different perfusionsolution for hypothermic perfusion, optionally the box 10 shown in FIG.2. The first perfusion solution may contain at least one oxygen carrier,may be oxygenated and/or may be selected from the group consisting of across-linked hemoglobin and stabilized red blood cells. The secondperfusion solution may be non-oxygenated, may contain at least oneanti-oxidant, and/or may contain at least one vasodilator. Additionally,the solution may contain no more than 5 mM of dissolved pyruvate salt.Also, the first container and the second container may be configured tobe operably connected to a perfusion machine as perfusion fluidreservoirs in fluid communication with perfusate conduits of saidperfusion machine. Further, one of the first and second containers maybe compressible to apply pressure to the perfusion solution therein.Furthermore, at least one of the first and second containers may includea first opening for passage of a contained perfusion solution out of thecontainer and a second opening passage of a compressed gas into thecontainer. The package may be a cassette configured to be operablyconnected to a perfusion machine for connection of the first and secondcontainers within the cassette in fluid communication with perfusateconduits or tubing of the perfusion machine.

In other embodiments, the perfusion solution kit may contain at leastone first container holding a first perfusion solution for hypothermicperfusion at a first temperature and at least one second containerholding a second, different perfusion solution for hypothermic perfusionat a second temperature lower than the first temperature. In the kit,the first perfusion solution may contain at least a crystalloid and maycontain at least one vasodilator. The second perfusion solution may beoxygen carrier enhanced, where the oxygen carrier is selected from thegroup consisting of a hemoglobin and stabilized red blood cells. Inaddition, the second perfusion solution may, if desired, contain atleast one anti-oxidant or free radical scavenger. Preferably, the saidsolution contains no more than 5 mM of dissolved pyruvate salt. Asabove, the first container and the second container may be configured tobe operably connected to a perfusion machine as perfusion fluidreservoirs in fluid communication with perfusate conduits of saidperfusion machine. Further, one of the first and second containers maybe compressible to apply pressure to the perfusion solution therein.Furthermore, at least one of the first and second containers may includea first opening for passage of a contained perfusion solution out of thecontainer and a second opening passage of a compressed gas into thecontainer. The package may be a cassette configured to be operablyconnected to a perfusion machine for connection of the first and secondcontainers within the cassette in fluid communication with perfusateconduits or tubing of the perfusion machine.

The medical fluid within reservoir 10 is preferably brought to apredetermined temperature by a first thermoelectric unit 30 a in heattransfer communication with the reservoir 10. A temperature sensor T3relays the temperature within the reservoir 10 to the microprocessor150, which adjusts the thermoelectric unit 30 a to maintain a desiredtemperature within the reservoir 10 and/or displays the temperature on acontrol and display areas 5 a for manual adjustment. Alternatively or inaddition, and preferably where the organ perfusion device is going to betransported, the medical fluid within the hypothermic perfusion fluidreservoir can be cooled utilizing a cryogenic fluid heat exchangerapparatus such as that disclosed in co-pending application Ser. No.09/039,443, which is hereby incorporated by reference.

An organ chamber 40 is provided which supports a cassette 65, as shownin FIG. 2, which holds an organ to be perfused, or a plurality ofcassettes 65,65,65, as shown in FIG. 12, preferably disposed oneadjacent the other. Various embodiments of the cassette 65 are shown inFIGS. 11A-11D. The cassette 65 is preferably formed of a material thatis light but durable so that the cassette 65 is highly portable. Thematerial may also be transparent to allow visual inspection of theorgan.

Preferably the cassette 65 includes side walls 67 a, a bottom wall 67 band an organ supporting surface 66, which is preferably formed of aporous or mesh material to allow fluids to pass therethrough. Thecassette 65 may also include a top 67 d and may be provided with anopening(s) 63 for tubing (see, for example, FIG. 11D). The opening(s) 63may include seals 63 a (e.g., septum seals or o-ring seals) andoptionally be provided with plugs (not shown) to prevent contaminationof the organ and maintain a sterile environment. Also, the cassette 65may be provided with a closeable air vent 61 (see, for example, FIG.11D). Additionally, the cassette 65 may be provided with tubing forconnection to the organ or to remove medical fluid from the organ bathand a connection device(s) 64 for connecting the tubing to, for example,tubing 50 c, 81, 82, 91 and/or 132 (see, for example, FIG. 11D). Thecassette 65, and more particularly the organ support, opening(s),tubing(s) and/or connection(s), may be specifically tailored to the typeof organ and/or size of organ to be perfused. Outer edges 67 c of theside support walls 67 a can be used to support the cassette 65 disposedin the organ chamber 40. The cassette 65 may further include a handleportion 68 which allows the cassette 65 to be easily handled, as shown,for example, in FIGS. 11C and 11D. Each cassette 65 may also be providedwith its own stepping motor/cam valve 65 (for example, in the handleportion 68, as shown in FIG. 11C) for fine tuning the pressure ofmedical fluid perfused into the organ 60 disposed therein, discussed inmore detail below. Alternatively, pressure may, in embodiments, becontrolled by way of a pneumatic chamber, such as individual pneumaticchamber for each organ (not shown), or by any suitable variable valvesuch as a rotary screw valve or a helical screw valve. The cassette 65is configured such that it may be removed from the organ perfusionapparatus 1 and transported to another organ perfusion apparatus in aportable transport apparatus, such as, for example, a conventionalcooler or a portable container such as that disclosed in simultaneouslyfiled co-pending U.S. application Ser. No. 09/161,919, which is herebyincorporated by reference.

When transported, the organ is disposed on the organ supporting surface66 and the cassette 65 is preferably enclosed in a preferably sterilebag 69, as shown, for example, in FIG. 11A. When the organ is perfusedwith medical fluid, effluent medical fluid collects in the bag 69 toform an organ bath. Alternatively, the cassette 65 can be formed with afluid tight lower portion in which the effluent medical fluid maycollect, or the effluent medical fluid may collect in the organ chamber40 to form the organ bath. In either alternative case, the bag 69 wouldpreferably be removed prior to inserting the cassette into the organchamber 40. Further, where a plurality of organs are to be perfused, anorgan chamber may be provided for each organ.

The organ bath is preferably cooled to a predetermined temperature by asecond thermoelectric unit 30 b in heat transfer communication with theorgan chamber 40. Alternatively and preferably where the organ perfusiondevice is going to be transported, the medical fluid within reservoir 10can be cooled utilizing a cryogenic fluid heat exchanger apparatus suchas that disclosed in co-pending application Ser. No. 09/039,443, whichis hereby incorporated by reference. A temperature sensor T2 within theorgan chamber 40 relays the temperature of the organ 60 to themicroprocessor 150, which adjusts the thermoelectric unit 30 b tomaintain a desired organ temperature and/or displays the temperature onthe control and display areas 5 c for manual adjustment.

Medical fluid may be fed from the bag 15 a directly to an organ 60disposed in the organ chamber 40 through tubing 50 a,50 b,50 c or frombag 15 b through tubing 50 d,50 e,50 c by opening valve LV₄ or LV₃,respectively. Conventional medical fluid bag and tubing connections areutilized. All tubing is disposable, easily replaceable andinterchangeable. Further, all tubing is formed of or coated withmaterials compatible with the medical fluids used, more preferablynon-thrombogenic materials. An end of the tubing 50 c is inserted intothe organ 60. The tubing is connected to the organ(s) with conventionalmethods, for example, with sutures. The tubing may include a lip tofacilitate connection to the organ. However, the specific methods andconnection depend on the type of organs(s) to be perfused.

The microprocessor 150 preferably controls the pressure source 20 inresponse to signals from the pressure sensor P1 to control the pressureof the medical fluid fed into the organ 60. The microprocessor 150 maydisplay the pressure on the control and display areas 5 a, optionallyfor manual adjustment. A fluid flow monitor F1 may also be provided onthe tubing 50 c to monitor the flow of medical fluid entering the organ60 to indicate, for example, whether there are any leaks present in theorgan.

Alternatively, the medical fluid may be fed from the reservoir tank 17via tubing 51 into an intermediary tank 70 preferably having a pressurehead of approximately 5 to 40 mm Hg. Medical fluid is then fed bygravity or, preferably, pressure, from the intermediary tank 70 to theorgan 60 along tubing 50 c by activating a valve LV₆. A level sensor 71is provided in the intermediary tank 70 in order to maintain thepressure head. Where a plurality of organ chambers 40 and organs 60 areprovided, the organs 60 are connected in parallel to the reservoir 10utilizing suitable tubing duplicative of that shown in FIG. 2. See, forexample, FIG. 12. The use of pneumatically pressurized and gravity fedfluid pumps configured to avoid overpressurization even in cases ofsystem failure prevents general tissue damage to the organ and thewashing away of or damage to the vascular endothelial lining of theorgan. Thus, organ perfusion in this system can be performed with eitherhydrostatic perfusion (gravity or pressure fed flow) or peristalticperfusion by introducing flow to the organ from a peristaltic (roller)pump.

A bubble detection system may be installed to sense bubbles in theperfusate. An air sensor and sensor board are preferably used. Theoutput of the sensor activates a debubbler system, such as an opensolenoid valve, to rid bubbles from the perfusate flow prior to organintroduction. As with all of the sensors and detectors in this system,the bubble detector may be positioned at any point in the system that iseffective based on the particular parameters or design characteristicsof the system. For example, a bubble detector and debubbler system BDmay be positioned between the cam valve 205 and pressure sensor P1, asshown in FIG. 1.

A stepping motor/cam valve 205, or other suitable variable valve such asa rotary screw valve, may be arranged on the tubing 50 c to providepulsatile delivery of the medical fluid to the organ 60, to decrease thepressure of the medical fluid fed into the organ 60, and/or to stop flowof medical fluid into the organ 60 if the perfusion pressure exceeds apredetermined amount. Specific embodiments of the stepping motor/camvalve are shown in FIGS. 13A-13B and 14A-14F. FIGS. 13A-13B show astepping motor/rotational type cam valve.

FIG. 13A is a top view of the apparatus. Tubing, for example, tubing 50c, is interposed between a support 203 and cam 200. Cam 200 is connectedby a rod 201 to stepping motor 202. FIG. 13B is a side view of theapparatus. The dashed line shows the rotational span of the cam 200. InFIG. 13B, the cam 200 is in its non-occluding position. Rotated 180degrees, the cam 200 totally occludes the tubing 50 c with varyingdegrees of occlusion therebetween. This stepping motor/cam valve isrelatively fast, for example, with respect to the embodiment shown inFIGS. 14A-14F; however, it requires a strong stepping motor.

FIGS. 14A-14F disclose another stepping motor/cam valve 210 according tothe invention. FIG. 14A is a side view of the apparatus while FIG. 14Cis a top view. Tubing, for example, tubing 50 c, is interposed betweencam 220 and support 223. The cam 220 is connected to stepping motor 222by supports 221 a-221 d and helical screw 225, which is connected to thestepping motor 222 via plate 222 a. FIG. 14B shows the supports 221 aand plate 222 a in front view. As show in FIG. 14D, where the support221 d is to the left of the center of the helical screw 225, the tubing50 c is not occluded. However, as the helical screw 225 is turned by thestepping motor 222, the support 221 d moves to the left (with respect toFIGS. 14D-14F) toward a position where the cam 220 partially or fullyoccludes the tubing 50 c. Such apparatus is slower than the apparatus ofFIGS. 13A-13B, but is more energy efficient.

Medical fluid expelled from the organ 60 which has collected in thebottom of the bag 69 (the cassette 65 or the organ chamber 40) is eitherpumped out through tubing 81 by a pump 80 for filtration, passingthrough a filter unit 82 and being returned to the organ bath, or ispumped out by a pump 90 for circulation through tubing 91. The pumps 80,90 are preferably conventional roller pumps; however, other types ofpumps may also be appropriate. A level sensor L2 in communication withthe microprocessor 150 ensures that a predetermined level of effluentmedical fluid is maintained within the organ chamber 40. A temperaturesensor T1 disposed in the tubing 91 relays the temperature of themedical fluid pumped out of the organ bath along tubing 91 to themicroprocessor 150, which monitors the same. A pressure sensor P2disposed along the tubing 91 relays the pressure therein to themicroprocessor 150, which shuts down the system if the fluid pressure inthe tubing 91 exceeds a predetermined limit, or activates an alarm tonotify the operator that the system should be shut down, for example, toclean filters or the like.

As the medical fluid is pumped along tubing 91 it passes through afilter unit 95 (e.g., 25μ, 8μ, 2μ, 0.8μ, 0.2μ and/or 0.1μ filters); aCO₂ scrubber/O₂ membrane 100 and an oxygenator 110, for example, aJOSTRA™ oxygenator. The CO₂ scrubber/O₂ membrane 100 is preferably ahydrophobic macroporous membrane with a hydrophilic (e.g., Hypol)coating in an enclosure. A vacuum source (not shown) is utilized toapply a low vacuum on a side opposite the hydrophilic coating by theactivation of valve VV₁. A hydrostatic pressure of approximately 100 mmHg is required for aqueous passage through the membrane. The mechanicalrelief valve (not shown) prevents the pressure differential fromattaining this level. Immobilized pegolated carbonic anhydrase may beincluded in the hydrophilic coating. This allows bicarbonate to beconverted to CO₂ and subsequently removed by vacuum venting. However,with organs such as kidneys which have the ability to eliminatebicarbonate, this may be unnecessary except in certain cases.

The oxygenator 110 is preferably a two stage oxygenator which preferablyincludes a hydrophilically coated low porosity oxygen permeablemembrane. A portion of the medical fluid is diverted around theoxygenator along tubing 111 in which is disposed a viability sensor V1,which senses fluid characteristics, such as organ resistance(pressure/flow), pH, pO₂, pCO₂, LDH, T/GST, Tprotein and/or fluorescenttagged copolymer, indicative of an organ's viability. The viabilitysensor V1 is in communication with the microprocessor 150 and allows theorgan's viability to be assessed either automatically or manually. Oneof two gases, preferably 100% oxygen and 95/5% oxygen/carbon dioxide, isplaced on the opposite side of the membrane depending on the pH level ofthe diverted medical fluid. Alternatively, another pump (not shown) maybe provided which pumps effluent medical fluid out of the organ chamber40 and through a viability sensor before returning it to the bath, orthe viability sensor can be placed on tubing 81 utilizing pump 80.

Alternative to the pump 90, filter unit 95, the CO₂ scrubber/O₂ membrane100 and/or the oxygenator 10, a modular combined pump, filtration,oxygenation and/or debubbler apparatus may be employed such as thatdescribed in detail in simultaneously filed co-pending U.S. patentapplication Ser. No. 09/039,318, which is hereby incorporated byreference. As shown in FIGS. 4-10, the apparatus 5001 is formed ofstackable modules. The apparatus 5001 is capable of pumping a fluidthrough a system as well as oxygenating, filtering and/or debubbling thefluid. The modules are each formed of a plurality of stackable supportmembers and are easily combinable to form a compact apparatus containingdesired components. Filtration, oxygenation and/or degassing membranesare disposed between the support members.

FIGS. 4-8 show various modules that may be stacked to form a combinedpump, filtration, oxygenation and/or debubbler apparatus, such as thecombined pump, filtration, oxygenation and debubbler apparatus 5001shown in FIGS. 9-10. As depicted in these figures, the combined pump,filtration, oxygenation and debubbler apparatus 5001 is preferablyformed of a plurality of stackable support members groupable to form oneor more modules.

Interposed between the plurality of stackable support member arefiltration, oxygenation and/or degassing membranes depending on aparticular user's needs. The filtration, oxygenation and/or degassingmembranes are preferably commercially available macro-reticularhydrophobic polymer membranes hydrophilically grafted in a commerciallyknown way, such as, for example, ethoxylation, to prevent proteindeprivation, enhance biocompatibility with, for example, blood and toreduce clotting tendencies. The filtration membrane(s) is preferablyhydrophilically grafted all the way through and preferably has aporosity (pore size) within a range of 15 to 35μ, more preferably 20 to30μ, to filter debris in a fluid, preferably without filtering outcellular or molecular components of the fluid. The degassing membrane(s)and oxygenation membrane(s) are hydrophilically surface treated tomaintain a liquid-gas boundary. The degassing membrane(s) andoxygenation membrane(s) preferably have a porosity of 15μ or less, morepreferably 10μ or less.

The modules may include a first pump module 5010, as shown in explodedview in FIG. 4; a filtration module 5020, as shown in exploded view inFIG. 5; an oxygenation module 5030, as shown in exploded view in FIG. 6;a debubbler module 5040, as shown in exploded view in FIG. 7; and asecond pump module 5050, as shown in exploded view in FIG. 8. The pumpmodules are each connected to a source of pump fluid and are actuatedeither manually or by the microprocessor. The support members arepreferably similarly shaped. For example, the support members may eachbe plate-shaped; however, other shapes may also be appropriate. As shownin FIG. 10, the support members are preferably removably connected byscrews or bolts 5065; however, other fasteners for assembling theapparatus may also be appropriate.

The first pump module 5010 preferably includes a first (end) supportmember 5011, a second support member 5012 with a cut-out center area5012 c, a diaphragm 5013 and a third support member 5014. The supportmembers of this module and each of the other modules are preferably thinand substantially flat (plate-like), and can be formed of anyappropriate material with adequate rigidity and preferably alsobiocompatibility. For example, various resins and metals may beacceptable. A preferred material is an acrylic/polycarbonate resin.

The first (end) support member 5011 is preferably solid and providessupport for the pump module 5010. The first (end) support member 5011preferably includes a domed-out cavity for receiving pump fluid such asair. Tubing 5011 t is provided to allow the pump fluid to enter the pumpmodule 5010. The diaphragm 5013 may be made of any suitable elastic andpreferably biocompatible material, and is preferably polyurethane. Thethird support member 5014 includes a domed-out fluid cavity 5014 d andtubing 5014 t for receiving fluid, such as, for example, blood or anartificial perfusate, into the cavity 5014 d of the pump module 5010.The first pump module, or any of the other modules, may also include aport 5014 p for sensors or the like. Preferably hemocompatibleanti-backflow valves serve to allow unidirectional flow through the pumpmodule 5010.

The filtration module 5020 preferably includes a filtration membrane5021 m which forms a boundary of cavity 5014 d, a first support member5022 with a cut-out center area 5022 c, a degassing membrane 5022 m andsecond and third support members 5023 and 5024. The filtration membrane5021 m is preferably a 25μ macro-reticular filtration membrane modifiedto enhance biocompatibility with, for example, blood and to reduceclotting tendencies (like the other supports, filters and membranes inthe device). The degassing membrane 5022 m is preferably a 0.2-3μmacro-reticular degassing membrane with a reverse flow aqueous pressuredifferential of at least 100 mmHg for CO₂ removal surface modified toenhance biocompatibility.

The first support 5022 includes tubing 5022 t for forwarding fluid intothe oxygenation module 30, or another adjacent module, if applicable,after it has passed through the filtration membrane 5021 m and along thedegassing membrane 5022 m. The second support member 5023 of thefiltration module 5020 includes a domed-out fluid cavity 5023 d andtubing 5023 t through which a vacuum may be applied to the cavity 5023 dto draw gas out of the fluid through degassing membrane 5022 m. Thefourth support member 5024 is preferably solid and provides support forthe filtration module 5020. The third support member can also includetubing 5024 t through which a vacuum may be applied to draw gas out ofthe fluid through the degassing membrane 5031 m of the oxygenationmodule 5030 as discussed below. The filtration module 5020, or any ofthe other modules, may also include a port 5023 p for sensors or thelike.

The oxygenation module 5030 includes a degassing membrane 5031 m, afirst support member 5032, a filtration membrane 5033 m, an oxygenationmembrane 5034 m, a second support member 5034 with a cut-out center area5034 c, and third and fourth support members 5035, 5036. The degassingmembrane 5031 m is preferably a 0.2-3μ macro-reticular degassingmembrane with a reverse flow aqueous pressure differential of at least100 mmHg surface modified to enhance biocompatibility.

The first support member 5032 includes a domed-out fluid cavity 5032 d.The surface of the domed-out fluid cavity 5032 d preferably forms atortuous path for the fluid, which enhances the oxygenation anddegassing of the fluid. The filtration membrane 5033 m is preferably a25μ macro-reticular filtration membrane modified to enhancebiocompatibility. The oxygenation membrane 5034 m is preferably a 0.2-1μmacro-reticular oxygenation membrane with a reverse flow aqueouspressure differential of at least 100 mmHg surface modified to enhancebiocompatibility.

The second support member 5034 includes tubing 5034 t for forwardingfluid out of the oxygenation module 5030 into the debubbler module 5040,or another adjacent module, if applicable. The third support member 5035includes a domed-out cavity 5035 d and tubing 5035 t for receivingoxygen from an external source. The fourth support member 5036 ispreferably solid and provides support for the oxygenation module 5030.

The debubbler module 5040 includes a first support member 5041, afiltration membrane 5042 m, a degassing membrane 5043 m, a secondsupport member 5043 having a cut-out center area 5043 c, and a thirdsupport member 5044. The first support member 5041 has a domed-out fluidcavity 5041 d.

The filtration membrane 5042 m is preferably a 25μ macro-reticularfiltration membrane modified to enhance biocompatibility. The degassingmembrane 5043 m is preferably a 0.2-3μ macro-reticular degassingmembrane with a reverse flow aqueous pressure differential of at least100 mmHg surface modified to enhance biocompatibility. The secondsupport member 5043 has tubing 5043 t for forwarding fluid out of thedebubbler module 5040 into the pump module 5050, or another adjacentmodule, if applicable. The third support member 5044 includes adomed-out cavity 5044 d and tubing 5044 t through which a vacuum may beapplied to draw gas out of the fluid through the degassing membrane 5043m.

The second pump module 5050 may correspond to the first pump module5010. It preferably includes a first support member 5051, a diaphragm5052, a second support member 5053 with a cut-out center area 5053 c,and a third (end) support member 5054. The first support member 5051includes a domed out fluid cavity 5051 d and tubing 5051 t for allowingfluid to exit the pump module. The diaphragm 5052 is preferably apolyurethane bladder.

The third (end) support piece member 5054 is preferably solid andprovides support for the pump module 5050. Support member 5054preferably includes a domed out cavity (not shown) for receiving pumpfluid. Tubing 5054 a is provided to allow the pump fluid such as air toenter the pump module 5050. Preferably hemocompatible anti-backflowvalves may serve to allow unidirectional flow through the pump module5050.

In operation, blood and/or medical fluid enters the first pump module5010 through tube 5014 t passes through the filtration membrane 5021 mand along the degassing membrane 5022 m. A small vacuum is appliedthrough tubing 5023 t to draw gas through the degassing membrane 5022 m.Next, the blood and/or medical fluid travels into the oxygenation module5030 via internal tubing 5022 t, passing along the degassing membrane5031 m, through the filtration membrane 5033 m and along the oxygenationmembrane 5034 m. Oxygen is received into the domed-out cavity 5035 d ofthe third support member of the oxygenation module 5030 via tubing 5035t and passes through the oxygenation membrane 5034 m into the bloodand/or medical fluid as the blood and/or medical fluid travels along itssurface.

After being oxygenated by the oxygenation module 5030, the blood and/ormedical fluid then travels via internal tubing 5034 t into the debubblermodule 5040. The blood and/or medical fluid passes through thefiltration membrane 5042 m and along the degassing membrane 5043 m. Asmall vacuum force is applied through tubing 5044 t to draw gas out ofthe blood and/or medical fluid through the degassing membrane 5043 m.After passing through the degassing module 5040, the blood and/ormedical fluid travels into the second pump module 5050 through tubing5041 t, and exits the second pump module 5050 via tubing 5051 t.

After passing through the oxygenator 110, or alternatively through thecombined pump, oxygenation, filtration and/or degassing apparatus 5001,the recirculated medical fluid is selectively either directed to thereservoir 15 a or 15 b not in use along tubing 92 a or 92 b,respectively, by activating the respective valve LV₂ and LV₅ on thetubing 92 a or 92 b, or into the organ chamber 40 to supplement theorgan bath by activating valve LV₁. Pressure sensors P3 and P4 monitorthe pressure of the medical fluid returned to the bag 15 a or 15 b notin use. A mechanical safety valve MV₂ is provided on tubing 91 to allowfor emergency manual cut off of flow therethrough. Also, tubing 96 andmanual valve MV₁ are provided to allow the apparatus to be drained afteruse and to operate under a single pass mode in which perfusate exitingthe organ is directed to waste rather than being recirculated(recirculation mode.)

A bicarbonate reservoir 130, syringe pump 131 and tubing 132, and anexcretion withdrawal unit 120, in communication with a vacuum (notshown) via vacuum valve VV₂, and tubing 121 a, 122 a are also eachprovided adjacent to and in communication with the organ chamber 40.

The method according to the invention preferably utilizes apparatus suchas that discussed above to perfuse an organ to sustain, monitor and/orrestore the viability of an organ and/or to transport and/or store theorgan. Preservation of the viability of an organ is a key factor to asuccessful organ transplant. Organs for transplant are often deprived ofoxygen (known as ischemia) for extended periods of time due to diseaseor injury to the donor body, during removal of the organ from the donorbody and/or during storage and/or transport to a donee body. The methodaccording to the present invention focuses on three concepts in order topreserve an organ's viability prior to transplant of the organ into adonor body—treating the cellular mitochondria to maintain and/or restorepre-ischemia energy and enzyme levels, preventing general tissue damageto the organ, and preventing the washing away of or damage to thevascular endothelial lining of the organ.

The mitochondria are the energy source in cells. They need large amountsof oxygen to function. When deprived of oxygen, their capacity toproduce energy is reduced or inhibited. Additionally, at temperaturesbelow 20° C. the mitochondria are unable to utilize oxygen to produceenergy. By perfusing the organ with an oxygen rich medical fluid atnormothermic temperatures, the mitochondria are provided with sufficientamounts of oxygen so that pre-ischemia levels of reserve high energynucleotide, that is, ATP levels, in the organ reduced by the lack ofoxygen are maintained and/or restored along with levels of enzymes thatprotect the organ's cells from free radical scavengers. Pyruvate richsolutions, such as that disclosed in U.S. Pat. No. 5,066,578, areincapable of maintaining and/or restoring an organ's pre-ischemia energylevels and only function in the short term to raise the level of ATP asmall amount. That is, organs naturally have significant pyruvatelevels. Providing an organ with additional pyruvate will not assist inrestoring and/or maintaining the organ's pre-ischemia energy levels ifthe mitochondria are not provided with sufficient oxygen to produceenergy. Thus, the normothermic perfusion fluid may contain pyruvate butmay also contain little or no pyruvate. For example, it can contain lessthan 6 mM of pyruvate, 5 mM, 4 mM, or even no pyruvate. Other knownpreservation solutions, such as that disclosed in U.S. Pat. No.5,599,659, also fail to contain sufficient oxygen to restore and/ormaintain pre-ischemia energy and enzyme levels.

After maintaining and/or restoring the organ's pre-ischemia energylevels by perfusing the organ with an oxygen rich first medical fluid atnormothermic or near-normothermic temperatures (the normothermic mode),the organ is perfused with a second medical fluid at hypothermictemperatures (the hypothermic mode). The hypothermic temperatures slowthe organ's metabolism and conserve energy during storage and/ortransport of the organ prior to introduction of the organ into a doneebody. The medical fluid utilized in the hypothermic mode contains littleor no oxygen, which cannot be utilized by mitochondria to produce energybelow approximately 20° C. The medical fluid may include antioxidantsand other tissue protecting agents, such as, for example, ascorbic acid,glutathione, water soluble vitamin E, catalase, or superoxide dismutaseto protect against high free radical formation which occurs at lowtemperatures due to the reduction in catalase/superoxide dismutaseproduction. Further, various drugs and agents such as hormones,vitamins, nutrients, antibiotics and others may be added to eithersolution where appropriate. Additionally, vasodilators, such as, forexample, peptides, may be added to the medical fluid to maintain floweven in condition of injury.

Prior to any normothermic perfusion with the oxygen rich first medicalfluid at normothermic temperatures, the organ may be flushed with amedical solution containing little or no oxygen and preferablycontaining antioxidants, anti-apoptic agents, and agents that decreasevascular permeability. The flushing is usually performed at hypothermictemperatures but can, if desired and/or as necessary, be performed atnormothermic or near-normothermic temperatures. Flushing or perfusionmay reduce or stop catabolic changes, such as free radical activity,apoptic enzymatic degradation and vascular permeability. Flushing can befollowed by one or more of hypothermic perfusion, normothermicperfusion, and/or static storage, in any necessary and/or desired order.In some cases, normothermic perfusion may not be necessary.

The normothermic perfusion, with or without prior hypothermic flushing,may also be performed on an organ that has already been subjected tohypothermic temperatures under static or perfusion conditions, as wellas on normothermic organs. A medical fluid under normothernic conditionsmay also include an oxygen carrier, a free radical scavenger, apituitary growth factor extract and cell culture media.

The organ may be perfused at normothermic or near-normothermictemperatures to sustain, monitor and/or restore its viability priorand/or subsequent to being perfused at hypothermic temperatures forstorage and then may be transported without or preferably withhypothermic perfusion. Also, the normothermic perfusion may be performedin vivo prior to removal of the organ from the donor body. Further, theorgan may be perfused at normothermic temperatures to sustain, monitorand/or restore its viability prior to being perfused at hypothermictemperatures preparatory to storage and/or transport. Then the organ maybe transplanted into a donee body while remaining at hypothermictemperatures, or it may first be subjected to normothermic perfusion tohelp it recover from the effects of storage and/or transport. In thelatter case, it may then be transplanted at normothermic temperatures,or preferably, be hypothermically perfused again for transplantation athypothermic temperatures. After transplant, the organ may optionallyagain be perfused at normothermic temperatures in vivo, or allowed towarm up from the circulation of the donee.

By way of Example only, and without being limited thereto, FIG. 16 showsan exemplary diagram of possible processing steps according to theinvention. The Figure shows various possible processing steps ofmultiple organ recovery (MOR) from organ explant from the organ donorthrough implant in the donee, including possible WIT (warm ischemiatime) and hypoxia damage assessment. Several exemplary scenarios are setforth in the following discussion.

For example, in one embodiment of the present invention, the organ canbe harvested from the donor under beating heart conditions. Followingharvesting, the organ can be flushed, such as with any suitable solutionor material including, but not limited to VIASPAN (a preservationsolution available from DuPont), other crystalloid solution, dextran,HES (hydroxyethyl starch), or the like. The organ can then be storedstatically, for example, at ice temperatures (for example of from about1 to about 10° C.).

In another embodiment, such as where the organ has minimal WIT andminimal vascular occlusion, a different procedure can be used. Here, theorgan can again be harvested under beating heart conditions, followed byflushing, preferably at hypothermic temperatures. If necessary totransport the organ, the organ can be stored in a suitable transporterat, for example, ice temperatures. Flow to the organ can be controlledby a set pressure maximum, where preset pressure minimum and pressuremaximum values control the pulse wave configuration. If necessary tostore the organ for a longer period of time, such as for greater than 24hours, the organ can be placed in the MOR. In the MOR, a suitableperfusate can be used, such as a crystalloid solution, dextran or thelike, and preferably at hypothermic temperatures. Preferably, thehypothermic temperatures are from about 4 to about 10° C., but higher orlower temperatures can be used, as desired and/or necessary. Preferably,the perfusate solution contains specific markers to allow for damageassessment, although damage assessment can also be made by other knownprocedures. When desired, the organ can then be returned to thetransporter for transport to the implant site.

As a variation of the above procedure, an organ having minimal WIT andminimal vascular occlusion can be harvested under non-beating heartconditions. Here, the organ can flushed, preferably at hypothermictemperatures and, if necessary, stored for transport in a suitabletransporter at, for example, ice temperatures. As above, flow to theorgan can be controlled by a set pressure maximum, where preset pressureminimum and pressure maximum values control the pulse waveconfiguration. The organ can be placed in the MOR, either for extendedstorage and/or for damage assessment. In the MOR, a suitable perfusatecan be used, such as a crystalloid solution, dextran or the like, andpreferably at hypothermic temperatures. Preferably, the hypothermictemperatures are from about 4 to about 10° C., but higher or lowertemperatures can be used, as desired and/or necessary. Preferably, theperfusate solution contains specific markers to allow for damageassessment, although damage assessment can also be made by other knownprocedures. Following hypothermic perfusion, a second perfusion can beutilized, preferably at normothermic temperatures. Any suitableperfusion solution can be used for this process, including solutionsthat contain, as desired, oxygenated media, nutrients, and/or growthfactors. Preferably, the normothermic temperatures are from about 10 toabout 24° C., and more preferably from about 12 to about 24° C., buthigher or lower temperatures can be used, as desired and/or necessary.The normothermic perfusion can be conducted for any suitable period oftime, for example, for from about 1 hour to about 24 hours. Followingrecovery from the normothermic perfusion, the organ is preferablyreturned to a hypothermic profusion using, for example, a suitablesolution such as a crystalloid solution, dextran or the like, andpreferably at hypothermic temperatures. When desired, the organ can thenbe returned to the transporter for transport to the implant site.

In embodiments where the organ has high WIT, and/or where there is ahigh likelihood of or actual; vascular occlusion, variations on theabove processes can be used. For example, in the case where the organ isharvested under non-beating heart conditions, the organ can be flushedas described above. In addition, however, free radical scavengers can beadded to the flush solution, if desired. As above, the organ can bestored for transport in a suitable transporter at, for example, icetemperatures, where flow to the organ can be controlled by a setpressure maximum, and where preset pressure minimum and pressure maximumvalues control the pulse wave configuration. The organ can be placed inthe MOR, either for extended storage and/or for damage assessment. Inthe MOR, a suitable perfusate can be used, such as a crystalloidsolution, dextran or the like, and preferably at hypothermictemperatures. Preferably, the hypothermic temperatures are from about 4to about 10° C., but higher or lower temperatures can be used, asdesired and/or necessary. Preferably, the perfusate solution containsspecific markers to allow for damage assessment, although damageassessment can also be made by other known procedures. Followinghypothermic perfusion, a second perfusion can be utilized, preferably atnormothermic temperatures. Any suitable perfusion solution can be usedfor this process, including solutions that contain, as desired,oxygenated media, nutrients, and/or growth factors. Preferably, thenormothermic temperatures are from about 10 to about 24° C., and morepreferably from about 12 to about 24° C., but higher or lowertemperatures can be used, as desired and/or necessary. The normothermicperfusion can be conducted for any suitable period of time, for example,for from about 1 hour to about 24 hours. If desired, and particularly inthe event that vascular occlusion is determined or assumed to bepresent, a further perfusion can be conducted at higher normothermictemperatures, for example of from about 24 to about 37° C. This furtherperfusion can be conducted using a suitable solution that contains adesired material to retard the vascular occlusion. Such materialsinclude, for example, clotbusters such as streptokinase. Followingrecovery from the normothermic perfusion(s), the organ is preferablyreturned to a hypothermic profusion using, for example, a suitablesolution such as a crystalloid solution, dextran or the like, andpreferably at hypothermic temperatures. When desired, the organ can thenbe returned to the transporter for transport to the implant site.

The organ cassette according to the present invention allows an organ(s)to be easily transported to an organ recipient and/or between organperfusion apparatus in a portable transport apparatus, such as, forexample, a conventional cooler or a portable container such as thatdisclosed in co-pending U.S. application Ser. No. 09/161,919. Becausethe organ cassette may be provided with openings to allow the insertionof tubing of an organ perfusion apparatus into the cassette forconnection to an organ disposed therein, or may be provided with its owntubing and connection device or devices to allow connection to tubingfrom an organ perfusion apparatus and/or also with its own valve, itprovides a protective environment for an organ for storage and/ortransport while facilitating insertion of the organ into and/orconnection of an organ to the tubing of an organ perfusion device.Further, the organ cassette may also include a handle to facilitatetransport of the cassette and may be formed of a transparent material sothe organ may be visually monitored.

Optionally, the apparatus may include a Global Positioning System (GPS)(not shown) to allow tracking of the location of the organ(s). Theapparatus may also include a data logger and/or transmitter (not shown)to allow monitoring of the organ(s) at the location of the apparatus orat another location.

The method of the invention will be discussed below in terms of theoperation of the apparatus shown in FIG. 2. However, other apparatus maybe used to perform the inventive method.

As previously discussed, the apparatus discussed above can operate intwo modes: a normothermic perfusion mode and a hypothermic perfusionmode. The normothermic perfusion mode will be discussed first followedby a discussion of hypothermic perfusion mode. Repetitive descriptionwill be omitted.

In the normothermic or near-normothermic perfusion mode, an organ isperfused for preferably ½ to 6 hours, more preferably ½ to 4 hours, mostpreferably ½ to 1 hour, with a medical fluid maintained preferablywithin a range of approximately 10° C. to 38° C., more preferably 12° C.to 35° C., even more preferably from about 10 to about 24° C., and mostpreferably 12° C. to 24° C. or 18° C. to 24° C. (for example, roomtemperature 22-23° C.) by the thermoelectric unit 30 a disposed in heatexchange communication with the medical fluid reservoir 10.

As discussed above, in this mode, the medical fluid is preferably anoxygenated cross-linked hemoglobin-based bicarbonate solution.Cross-linked hemoglobin-based medical fluids can deliver up to 150 timesmore oxygen to an organ per perfusate volume than, for example, a simpleUniversity of Wisconsin (UW) gluconate type perfusate. This allowsnormothermic perfusion for one to two hours to partially or totallyrestore depleted ATP levels. However, the invention is not limited tothis preservation solution. Other preservation solutions, such as thosedisclosed in U.S. Pat. Nos. 5,149,321, 5,234,405 and 5,395,314 andco-pending U.S. patent application Ser. No. 08/484,601, which are herebyincorporated by reference, may also be appropriate.

In the normothermic perfusion mode, the medical fluid is fed directly toan organ disposed within the organ chamber 40 from one or the other ofbags 15 a, 15 b via tubing 50 a,50 b,50 c or 50 d,50 e,50 c,respectively. The organ is perfused at flow rates preferably within arange of approximately 3 to 5 ml/gram/min. Pressure sensor P1 relays theperfusion pressure to the microprocessor 150, which varies the pressuresupplied by the pressure source 20 to control the perfusion pressureand/or displays the pressure on the control and display areas 5 a formanual adjustment. The pressure is preferably controlled within a rangeof approximately 10 to 100 mm Hg, preferably 50 to 90 mm Hg, by thecombination of the pressure source 20 and pressure cuff 15 a, 15 b inuse and the stepping motor/cam valve 65. The compressor and cuffsprovide gross pressure control. The stepping motor/cam valve 65 (orother variable valve or pressure regulator), which is also controlled bythe operator, or by the microprocessor 150 in response to signals fromthe pressure sensor P1, further reduces and fine tunes the pressureand/or puts a pulse wave on the flow into the organ 60. If the perfusionpressure exceeds a predetermined limit, the stepping motor/cam valve 65may be activated to shut off fluid flow to the organ 60.

The specific pressures, flow rates and length of perfusion time at theparticular temperatures will vary depending on the particular organ ororgans being perfused. For example, hearts and kidneys are preferablyperfused at a pressure of approximately 10 to 100 mm Hg and a flow rateof approximately 3 to 5 ml/gram/min. for up to approximately 2 to 4hours at normothermic temperatures to maintain and/or restore theviability of the organ by restoring and/or maintaining pre-ischemiaenergy levels of the organ, and are then preferably perfused at apressure of approximately 10 to 30 mm Hg and a flow rate ofapproximately 1 to 2 ml/gram/min. for as long as approximately 72 hoursto 7 days at hypothermic temperatures for storage and/or transport.However, these criteria will vary depending on the condition of theparticular organ, the donor body and/or the donee body and/or on thesize of the particular organ. One of ordinary skill in the art canselect appropriate conditions without undue experimentation in view ofthe guidance set forth herein.

Effluent medical fluid collects in the bottom of the organ chamber 40and is maintained within the stated temperature range by the secondthermoelectric unit 30 b. The temperature sensor T2 relays the organtemperature to the microprocessor 150, which controls the thermoelectricunit 30 a to adjust the temperature of the medical fluid and organ bathto maintain the organ 60 at the desired temperature, and/or displays thetemperature on the control and display areas 5 c for manual adjustment.

Collected effluent medical fluid is pumped out by the pump 80 via tubing81 through the filter unit 82 and then returned to the organ bath. Thisfilters out surgical and/or cellular debris from the effluent medicalfluid and then returns filtered medical fluid to act as the bath for theorgan 60. Once the level sensor L2 senses that a predetermined level ofeffluent medical fluid is present in the organ chamber 40 (preferablyenough to maintain the organ 60 immersed in effluent medical fluid),additional effluent medical fluid is pumped out by the pump 90 throughtubing 91. The temperature sensor T1 relays the temperature of the organbath to the microprocessor 150, which controls the thermoelectric unit30 b to adjust the temperature of the medical fluid to maintain theorgan 60 at the desired temperature and/or displays the temperature onthe control and display area 5 c for manual adjustment and monitoring.

As noted above, the medical fluid can be directed to waste in a singlepass mode or recirculated eventually back to the organ and/or bath(recirculation mode.)

Along tubing 91, the recirculated medical fluid is first pumped throughthe filter unit 95. Use of a cross-linked hemoglobin medical fluidallows the use of sub-micron filtration to remove large surgical debrisand cellular debris, as well as bacteria. This allows the use of minimalantibiotic levels, aiding in preventing organ damage such as renaldamage.

Next, the recirculated medical fluid is pumped through the CO₂scrubber/O₂ membrane 100. The medical fluid passes over the hydrophobicmacroporous membrane with a hydrophilic coating (for example, Hypol) anda low vacuum is applied on the opposite side by activating valve VV₁which removes CO₂ from the recirculated medical fluid.

Subsequently, a portion of the medical fluid then enters the oxygenator110 (for example, a JOSTRA™ oxygenator) and a portion is divertedtherearound passing via tubing 111 though the pH, pO₂, pCO₂, LDH, T/GST,Tprotein, and fluorescent tagged copolymer sensor V1. At this point twogases, preferably 100% oxygen and 95/5% oxygen/carbon dioxide, arerespectively placed on the opposite sides of the membrane depending onthe pH level of the diverted medical fluid. The gases are applied at apressure of up to 200 mm Hg, preferably 50 to 100 mm Hg, preferablythrough a micrometer gas valve GV₃. The cross-linked hemoglobin-basedbicarbonate medical fluid may be formulated to require a pCO₂ ofapproximately 40 mm Hg to be at the mid point (7.35) of a preferred pHrange of 7.25-7.45.

If the medical fluid exiting the oxygenator is within the preferred pHrange (e.g., 7.25-7.45), 100% oxygen is delivered to the gas exchangechamber, and valve LV₁ is then not opened, allowing the perfusate toreturn to the reservoir 10 into the bag 15 a or 15 b not in use. If thereturning perfusate pH is outside the range on the acidic side (e.g.,less than 7.25), 100% oxygen is delivered to the gas exchange chamberand valve LV₁ is then opened allowing the perfusate to return to theorgan chamber 40. Actuation of syringe pump 131 pumps, for example, onecc of a bicarbonate solution from the bicarbonate reservoir 130, viatubing 132 into the organ bath. Medical fluids with high hemoglobincontent provide significant buffering capacity. The addition ofbicarbonate aids in buffering capacity and providing a reversible pHcontrol mechanism.

If the returning perfusate pH is outside the range on the basic side(e.g., greater than 7.25), 95/5% oxygen/carbon dioxide is delivered tothe gas exchange chamber and valve LV₁ is not actuated, allowing theperfusate to return to the bag 15 a or 15 b not in use. The bag 15 a or15 b not in use is allowed to degas (e.g., any excess oxygen) throughvalve GV₄. When the bag 15 a or 15 b in use has approximately 250 ml orless of medical fluid remaining therein, its respective cuff 16 a, 16 bis allowed to vent via its respective gas valve GV₁, GV₂. Then, therespective cuff 16 a, 16 b of the bag 15 a or 15 b previously not in useis supplied with gas from the compressed gas source 20 to delivermedical fluid to the organ to continue perfusion of the organ.

In the hypothermic mode, an organ is perfused with a cooled medicalfluid, preferably at a temperature within a range of approximately 1° C.to 15° C., more preferably 4° C. to 10° C., most preferably around 10°C. The medical fluid is preferably a crystalloid perfusate withoutoxygenation and preferably supplemented with antioxidants and othertissue protecting agents, such as, for example, ascorbic acid,glutathione, water soluble vitamin E, catalase, or superoxide dismutase.

Instead of feeding the medical fluid directly to the organ, the medicalfluid may be fed from the reservoir tank 17 via tubing 51 into anintermediary tank 70 preferably having a pressure head of approximately5 to 40 mm Hg, more preferably 10 to 30 mm Hg, most preferably around 20mm Hg. Medical fluid is then fed by gravity or, preferably, pressure,from the intermediary tank 70 to the organ 60 along tubing 50 c byactivating a valve LV₆. The level sensor 71 in the intermediary tank 70is used to control the feed from reservoir tank 17 to maintain thedesired pressure head. Because the medical fluid is fed to the organ bygravity or, preferably, pressure, in the hypothermic mode, there is lessperfusion pressure induced damage to the delicate microvasculature ofthe organ. In fact, the pressure at which the organ is perfused islimited by the pressure head to at most 40 mm Hg.

The stepping motor/cam valve 205 (or other variable valve or pressureregulator) may be arranged on the tubing 50 c to provide pulsatiledelivery of the medical fluid to the organ 60, to decrease the pressureof the medical fluid fed into the organ 60 for control purposes, or tostop flow of medical fluid into the organ 60, as described above.

Further, in the hypothermic mode, because the organ 60 has less of ademand for nutrients, the medical fluid may be provided to the organ 60intermittently (e.g., every two hours at a flow rate of up toapproximately 100 ml/min.), or at a slow continuous flow rate (e.g., upto approximately 100 ml/min.) over a long period of time. Intermittentperfusion can be implemented in the single pass mode or recirculationmode. The pump 80, filter unit 82 and tube 81 may be used to filter theorgan bath along with use of the pH, pO₂, pCO₂, LDH, T/GST, Tprotein,and fluorescent tagged copolymer sensor; however, because the organ isunable to utilize oxygen at hypothermic temperatures, the oxygenator isnot used. If desired and/or necessary, adequate oxygen can be obtainedfrom filtered room air or other suitable source.

Both the perfusate flow and the temperature regulation can beautomatically controlled. Such automatic control allows a rapid andreliable response to perfusion conditions during operation. Automaticflow control can be based on the parameters measured from the system,including the perfusate flow rate, the perfusate pH exiting the organ,the organ inlet pressure or timed sequences such as pre-selected flowrates or switching between perfusate modes. Preferably, the flow controlis based on pressure monitoring of the perfusate inflow into the organ.The benefits of automatic flow control include maintaining properoxygenation and pH control while operating under continuous flow orcontrolled intermittent flow. Thermal control of the thermoelectricdevices (TED) can regulate the temperature of the organ cassette orcontainer and the perfusate reservoir. The thermal control is based onthermal measurements made for example by thermistor probes in theperfusate solution or inside the organ or by sensors in the TED.

The automatic control is preferably effected by an interactive controlprogram using easily operated menu icons and displays. The parametersmay be prestored for selection by a user or programmed by the userduring operation of the system. The control program is preferablyimplemented on a programmed general purpose computer. However, thecontroller can also be implemented on a special purpose computer, aprogrammed microprocessor or microcontroller and peripheral integratedcircuit elements, an ASIC or other integrated circuit, a digital signalprocessor, a hardwired electronic or logic circuit such as a discreteelement circuit, a programmable logic device such as a PLD, PLA, FPGA orPAL, or the like. In general, any device capable of implementing afinite state machine that is in turn capable of implementing the controlprocess described herein may be used. The control program is preferablyimplemented using a ROM. However, it may also be implemented using aPROM, an EPROM, an EEPROM, an optical ROM disk, such as a CD-ROM orDVD-ROM, and disk drive or the like. However, if desired, the controlprogram may be employed using static or dynamic RAM. It may also beimplemented using a floppy disk and disk drive, a writable optical diskand disk drive, a hard drive, flash memory or the like.

In operation, as seen in FIG. 15, the basic steps of operation tocontrol perfusion of one or more organs include first inputting organdata. The organ data includes at least the type of organ and the mass.Then, the program will prompt the user to select one or more types ofperfusion modes. The types of perfusion modes, discussed above, includehypothermic perfusion, normothermic perfusion, and sequential perfusionusing both normothermic and hypothermic perfusion. When bothnormothermic and hypothermic perfusion are employed, the user can selectbetween medical fluids at different temperatures. Of course, the systemincludes default values based on previously stored values appropriatefor the particular organ. The user may also select intermittentperfusion, single pass perfusion, and recirculation perfusion. Dependingon the type of perfusion selected, aerobic or anaerobic medical fluidsmay be specified.

Next, the type of flow control for each selected perfusion mode is set.The flow control selector selects flow control based on at least one ofperfusate flow rate, perfusate pH, organ inlet pressure and timedsequences. In the preferred embodiment, the flow control is based ondetected pressure at the perfusion inlet to the organ. The flow of themedical fluid is then based on the selected perfusion mode and flowcontrol.

uring operation the conditions experienced by the system, in particularby the organ and the perfusate, are detected and monitored. The detectedoperating conditions are compared with prestored operating conditions. Asignal can then be generated indicative of organ viability based on thecomparison. The various detectors, sensors and monitoring devices aredescribed above, but include at least a pressure sensor, a pH detector,an oxygen sensor and a flow meter.

he control system may also include a thermal controller for controllingtemperature of at least one of the perfusate and the organ. The thermalcontroller can control the temperature of the medical fluid reservoirsand the organ container by controlling the TEDs. As noted above,temperature sensors are connected to the controller to facilitatemonitoring and control.

The control system may be manually adjusted at any time or set to followdefault settings. The system includes a logic circuit to prevent theoperator from setting parameters that would compromise the organ'sviability. As noted above, the system may also be operated in a manualmode for sequential hypothermic and/or normothermic perfusion, as wellas in the computer controlled mode for sequential hypothermic and/ornormothermic perfusion.

he above described apparatus and method may be used for child or smallorgans as well as for large or adult organs with modification as neededof the cassettes and or of the pressures and flow rates accordingly. Aspreviously discussed, the organ cassette(s) can be configured to theshapes and sizes of specific organs or organ sizes. The apparatus andmethod can also be used to provide an artificial blood supply to, such,for example, artificial placentas cell cultures, for growing/cloningorgan(s).

While the invention has been described in conjunction with a specificembodiment thereof, it is evident that many alternatives, modificationsand variations may be apparent to those skilled in the art. Accordingly,the preferred embodiment of the invention as set forth herein isintended to be illustrative, not limiting. Various changes may be madewithout departing from the spirit and scope of the invention as definedin the following claims.

1. A method of at least one of maintaining and restoring the viabilityof at least one organ subjected to a period of ischemia or hypoxia,comprising: perfusing said at least one organ with a first medical fluidat a first temperature to at least one of maintain and restorepre-ischemia or pre-hypoxia energy levels in the organ; and perfusingthe organ with a second medical fluid containing substantially no oxygenat a second temperature to at least one of store and transport theorgan, wherein said second temperature is lower than said firsttemperature.
 2. The method of claim 1, wherein the first temperature isup to about 25° C.
 3. The method of claim 1, wherein the firsttemperature is from about 12° C. to about 24° C.
 4. The method of claim1, wherein the first temperature is at least about 15° C.
 5. The methodof claim 1, wherein the first temperature is about room temperature. 6.The method of claim 1, wherein the first temperature is from about 20°C. to about 25° C.
 7. The method of claim 1, wherein the firsttemperature is from about 20° C. to about 38° C.
 8. The method of claim1, wherein the first temperature is from about 20° C. to about 35° C. 9.The method of claim 1, wherein the second temperature is from about 1°C. to about 15° C.
 10. The method of claim 1, wherein the secondtemperature is from about 4° C. to about 10° C.
 11. The method of claim1, wherein the first temperature is from about 12° C. to about 24° C.and the second temperature is at most 15° C.
 12. The method of claim 1,wherein the first temperature is at least about 20° C. and the secondtemperature is at most 15° C.
 13. The method of claim 1, wherein thesecond temperature is from about 4° C. to about 10° C.
 14. The method ofclaim 12, wherein the second temperature is from about 4° C. to about10° C.
 15. The method of claim 1, wherein the first medical fluid is anoxygenated solution.
 16. The method of claim 15, wherein the firstmedical fluid is an oxygenated hemoglobin-based solution and the secondfluid is a simple crystalloid solution augmented with antioxidants. 17.The method of claim 1, wherein the second medical fluid comprises atleast one member selected from the group consisting of an oxygencarrier, a free radical scavenger, a pituitary growth factor extract andcell culture media.
 18. The method of claim 17, wherein the firstmedical fluid comprises at least one viability marker.
 19. The method ofclaim 1, wherein the first and second fluids are the same medical fluid.20. A method of at least one of maintaining and restoring the viabilityof at least one organ subjected to a period of ischemia or hypoxia,comprising: perfusing said at least one organ with a medical fluid at afirst temperature to at least one of maintain and restore pre-ischemiaor pre-hypoxia energy levels in the organ the first temperature is fromabout 20° C. to about 24° C.; and perfusing the organ with the medicalfluid at a second temperature to at least one of store and transport theorgan, wherein said second temperature is lower than said firsttemperature, wherein the second temperature is from about 4° C. to about10° C.